Project Summary/Abstract Acute hyperglycemia is a common clinical problem in ischemic stroke patients, and is frequently associated with impaired thrombolytic efficacy of tPA and predictive of poor functional outcome of stroke patients. However, the clinical management of acute hyperglycemia remains uncertain. Recent studies showed that acute hyperglycemia is associated with rapid alteration in protein glycosylation, which is implicated in a wide range of extracellular signaling pathways and biological functions. It is thus possible that acute hyperglycemia dependent protein glycosylation might play a critical role in regulating thrombolysis and neurovascular injury in stroke patients. We propose to use clinical proteomic approaches to examine protein glycosylation in these patients and then probe mechanism in cell culture. The first aim is to systematically identify changes in blood protein glycosylation associated with acute hyperglycemia in tPA-treated stroke patients using MS-based approach to understand a broad landscape of clinical glyco-protein signals. Then, we validate the results in blood samples of an independent, large cohort of ischemic stroke patients, to select novel biomarkers with high translational impact. Finally, as brain microvascular endothelial cells play a critical role in regulating thrombolysis and blood vessel function via synthesizing and secreting regulatory factors, we aim to examine the glycosylation of proteins secreted by brain endothelial cells under hyperglycemic condition. This may help to understand the role of brain endothelium in regulating extracellular response to ischemic stroke related hyperglycemia. In this resubmission, we have revised extensively according to reviewers? comments, including adding new experts, new data and publications and experimental details and alternatives.